T-cells play critical roles in host immune responses to pathogens. However, they are also involved in allograft rejection and autoimmune responses. Understanding the mechanism by which the responses of cells are initiated is of fundamental importance towards their therapeutic manipulation. As a result of antigen recognition, the T-cell antigen receptor(TCR) induces as cascade of events that leads to the activation of these quiescent cells. The induction of protein tyrosine phosphorylation of critical intracellular substrates is the means by which the TCR initiates cellular responses. The TCR interacts directly with members of two families of protein tyrosine kinases (PTKs), the Src and Syk/ZAP-70 families, and regulates their functions. These PTKs are responsible, directly or indirectly, for the phosphorylation of a large number of intracellular proteins. Protein tyrosine phosphatases (PTPases), in turn, serve to regulate the basal and stimulated state of tyrosine phosphorylation. Genetic and biochemical data suggest that the CD45 PTPase plays a critical role in TCR signal transduction, probably by regulating the phosphorylation status of a tyrosine, Y505, which plays a negative regulatory role in Lck. The function and regulation of transmembrane PTPases, such as CD45 is not well understood. The overall goal of these studies is to understand how the structure of CD45 relates to its function and to its interaction with Lck. To achieve this goal, the crystal structure of the CD45 cytoplasmic domain will be solved in order to gain clues as to how the unusual structure of the tandem PTPase domains of CD45 relates to their functions. Using these structural clues as well as clues from biochemical studies, mutagenesis will be used to define the functions and mechanism of regulation of the tandem PTPase domains of CD45. New assays using fibroblast transformation and membrane biochemistry will be used to study the interaction of CD45 with its substrate, Lck, and the regulation of CD45 function by dimerization. These studies should provide insights into the function of CD45 as well as establish useful paradigms for the study of other transmembrane PTPases expressed in and outside of the hematopoietic lineage.